In Vivo Electrochemical Monitoring of the Change of Cochlear

Article pubs.acs.org/ac

In Vivo Electrochemical Monitoring of the Change of Cochlear Perilymph Ascorbate during Salicylate-Induced Tinnitus Junxiu Liu,† Ping Yu,‡ Yuqing Lin,‡ Na Zhou,† Tao Li,† Furong Ma,*,† and Lanqun Mao*,‡ †

Department of Otorhinolaryngology, Peking University Third Hospital, Beijing 100083, P. R. China Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences (CAS), Beijing 100190, P. R. China

‡

ABSTRACT: As one of the most important neurochemicals in biological systems, ascorbate plays vital roles in many physiological and pathological processes. In order to understand the roles of ascorbate in the pathological process of tinnitus, this study demonstrates an in vivo method for real time monitoring of the changes of ascorbate level in the cochlear perilymph of guinea pigs during the acute period of tinnitus induced by local microinfusion of salicylate with carbon fiber microelectrodes (CFMEs) modified with multiwalled carbon nanotubes (MWNTs). To accomplish in vivo electrochemical monitoring of ascorbate in the microenvironment of the cochlear perilymph, the MWNT-modified CFME is used as working electrode, a microsized Ag/AgCl is used as reference electrode, and Pt wire is used as counter electrode. Three electrodes are combined together around a capillary to form integrated capillary-electrodes. The integrated capillary-electrode is carefully implanted into the cochlear perilymph of guinea pigs and used both for externally microinfusing of salicylate into the cochlear perilymph and for real time monitoring of the change of ascorbate levels. The in vivo voltammetric method based on the integrated capillaryelectrodes possesses a high selectivity and a good linearity for ascorbate determination in the cochlear perilymph of guinea pigs. With such a method, the basal level of cochlear perilymph ascorbate is determined to be 45.0 ± 5.1 μM (n = 6). The microinfusion of 10 mM salicylate (1 μL/min, 5 min) into the cochlear decreases the ascorbate level to 28 ± 10% of the basal level (n = 6) with a statistical significance (P < 0.05), implying that the decrease in ascorbate level in the cochlear may be associated with salicylate-induced tinnitus. This study essentially offers a new method for in vivo monitoring of the cochlear perilymph ascorbate following the salicylate-induced tinnitus and can thus be useful for investigation on chemical essences involved in tinnitus.

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ischemia injury; loss of ascorbate and other low-molecularweight antioxidants during ischemia or other injury leaves cells vulnerable to oxidative damage.7 In addition to its function as an antioxidant and oxygen-radical scavenger, ascorbate has been considered as a neuromodulator for glutamate-mediated neurotransmission.8 On the other hand, although recent studies have established that tinnitus mechanisms involve both peripheral and central components of the auditory system, cochlea damage often initiates tinnitus and plays a key role in tinnitus generation.9 Furthermore, more and more evidence has suggested that hypoxia and ischemia are essential factors in the pathogenesis of tinnitus and that the 300 mg/kg dose of salicylate can decrease cochlear blood flow by about 25%.10 The important roles, which hypoxia/ischemia may have to play in the cochlea, substantially underline the importance of the role of the antioxidants in the pathogenesis of tinnitus.11 In addition, the increasing number of patients suffering from tinnitus could be explained by a dysbalance in the antioxidant defense system and ROS formation.12 While these demon-

nderstanding the chemical essences involved in the pathogenesis and therapeutics of tinnitus is of great importance because of the wide involvement of chemical processes both in the pathophysiology and therapeutics of inner diseases.1 For instance, it has been demonstrated that one of the major causes of human idiopathic sudden sensorineural hearing loss is impaired by low blood flow and oxygen delivery to the cochlea.2 Massive glutamate efflux in the perilymph of the cochlea is observed during cochlear ischemia. The excessive efflux of glutamate from inner hair cells results in cochlear excitotoxicity and often induces temporary hearing threshold shifts.3 Moreover, as one of the most important inner diseases, tinnitus is one of the widespread impairment of the hearing system.4 It is experienced by up to 15% of the general population. In the United States, approximately 10 million individuals seek medical attention among 40 million individuals with tinnitus.5 Despite the prevalence of tinnitus, the chemical essence involved in the pathophysiology of this disorder remains to be fully investigated. As one kind of the most important neurochemicals, ascorbate occurs physiologically as a water-soluble small molecule that has been demonstrated to play critical roles in many physiological and pathological processes.6 For example, it has been demonstrated to be neuroprotective toward cerebral © 2012 American Chemical Society

Received: April 25, 2012 Accepted: May 28, 2012 Published: May 28, 2012 5433

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strations strongly suggest that the knowledge on the change of the cochlear perilymph ascorbate level is very essential to understanding the chemical essence of tinnitus, the measurement of cochlear perilymph ascorbate remains challenging mainly because of the small volume of the cochlear perilymph and the risk of cerebral spinal fluid contamination in experimental animals as well as the chemical instability of ascorbate in the cochlear perilymph.13 In our earlier studies, we have demonstrated that the uses of carbon nanotubes as electrode materials can remarkably accelerate the electron transfer of the electrochemical oxidation of ascorbate, which eventually enable in vivo measurements of ascorbate in rat brain with a high selectivity and a good reproducibility.14 To validate this mechanism for in vivo monitoring of cochlear ascorbate during the acute period of salicylate-induced tinnitus in this study, we combine the electrodes (i.e., working, reference, and auxiliary electrodes) around a glassy capillary to form integrated capillary-electrodes. The capillary-electrodes are carefully implanted into the cochlear perilymph of guinea pigs and used both for externally microinfusing of salicylate into the cochlear perilymph of guinea pigs to establish tinnitus models and for real time monitoring of the change in ascorbate level following the models. The method demonstrated here is technically simple and reliable for in vivo monitoring of the change of ascorbate level in the cochlear perilymph and would find interesting applications in investigation of chemical essence involved in tinnitus.

Scheme 1. Schematic Illustration of Experimental Setup for in Vivo Monitoring of Cochlear Perilymph Ascorbate during Salicylate-Induced Tinnitus with Integrated CapillaryElectrodes

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under ambient temperature, and rinsed with distilled water. To prepare a microsized Ag/AgCl reference electrode for in vivo measurements, Ag wire (300 μm diameter) was polarized at +0.60 V (vs Ag/AgCl) in 0.10 M hydrochloride acid for 30 min to produce an Ag/AgCl wire. The MWNT-modified CFME, the microsized Ag/AgCl wire, and Pt wire (200 μm diameter) were carefully attached around the capillary with epoxy resin with 1:1 ethylendiamine as the harder under a microscope, with a close attention to well separate the electrodes from each other. Animals and Surgery. Male guinea pigs (weighing 350− 400 g) purchased from the Experimental Animal Center of Peking University were served as subjects. The guinea pigs were housed individually with food and water and maintained with a 12 h light/dark cycle and were randomly divided into an artificial perilymph microinfusion group (n = 6) and a salicylate microinfusion group (n = 6). After being anesthetized with pentobarbital sodium (400 mg/kg i.p.), the guinea pigs were operated by a retro-auricular approach to expose the cochlea. After the exposure of the cochlear basal turn, the mucosa was removed from the surface of the cochlea and the bone was allowed to dry. A small hole was drilled into the bone overlying scala tympani with a 0.25-mm steel drill at a distance of 1.5 mm from the round window. The implantation site for the capillaryelectrodes in the basal turn of scala tympani was coated with a thin layer of cyanoacrylate glue (Aesculap, Tuttlingen, Germany). After the implantation of the capillary-electrodes, cyanoacrylate glue was applied again followed by the application of a thin layer of two part silicone adhesive (KWIK-Cast silicone elastomer sealant, WPI, Sarasota) onto the dry cyanoacrylate glue layer. During the surgery and the electrochemical measurements, the body temperature of the animals was maintained at 37−38 °C with a heating pad and anesthetic was supplemented if necessary. In Vivo Monitoring of Perilymph Ascorbate and Electrocochleography. Electrochemical measurements were

EXPERIMENTAL SECTION Chemicals and Materials. Sodium ascorbate, salicylate, and ascorbate oxidase (AAox) (Cucurbita species, EC 1.10.3.3) were purchased from Sigma and used as supplied. Other chemicals were of analytical grade or higher and used as received. Stock solution of ascorbate (1.0 mM) was prepared just before use. Artificial perilymph was prepared by mixing NaCl (137 mM), KCl (5 mM), CaCl2 (2 mM), MgCl2 (1 mM), NaHCO3 (1 mM), and glucose (11 mM) into doubly distilled water, and the solution pH was adjusted to pH 7.4. Multiwalled carbon nanotubes (MWNTs, 10−30 nm in diameter and 0.5−50 μm in length) were purchased from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China). Prior to use, MWNTs were purified by refluxing the as-received MWNTs in 2.6 M nitric acid for 5 h. All aqueous solutions were prepared with doubly distilled water. Preparation of Integrated Capillary-Electrodes. To enable the synchronous occurrence of microinfusion and in vivo electrochemical measurements in the microenvironment of the cochlea perilymph of guinea pigs, the electrodes were integrated onto a fused-silica capillary (75-μm i.d., 150-μm o.d., Yongnian Optical Fiber Co., Hebei, China) to form integrated capillary-electrodes that were used both for microinfusing salicylate to establish an animal model of tinnitus and for in vivo real time monitoring of perilymph ascorbate (Scheme 1). Carbon fiber microelectrodes (CFMEs) were used as the substrate of a working electrode. The CFMEs were prepared by attaching a single carbon fiber (7 μm diameter) onto a copper wire with silver epoxy and the length of the fiber was trimmed to be about 100 μm under a microscope. To achieve the selectivity for the measurement of ascorbate, the CFMEs were modified with MWNTs by immersing the electrodes into 1 mg mL−1 MWNT dispersion into N,N-dimethylformamide. After 5 min, the electrodes were taken out from the dispersion, dried 5434

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performed with a computer-controlled electrochemical analyzer (CHI660B, Shanghai, China) with the as-prepared integrated capillary-electrodes for both in vitro and in vivo electrochemical measurements. The capillary was used to exogenously microinfuse the aqueous solutions of salicylate or AAox into the cochlear of guinea pigs. These solutions were delivered from gas-impermeable syringes and pumped through tetrafluoroethylene hexafluoropropene tubing by a microinjection pump (CMA 100, CMA Microdialysis AB, Stockholm, Sweden). The MWNT-modified CFME was polarized at +0.05 V for in vivo voltammetric monitoring of perilymph ascorbate. The experimental setup synchronously for microinfusion of salicylate and in vivo monitoring of perilymph ascorbate was schematically shown in Scheme 1. Electrocochleography recordings were performed by a computer and a DT3010/32 data acquisition board (data translation). Auditory evoked responses were recorded with a silver ball electrode on the niche of the round window, a reference electrode in the muscles of the neck, and a ground electrode in the Apex nasi. The cochlear microphonics (CM) were elicited by tone bursts at 4 kHz from 50 to 100 dB normal hearing level (nHL), with rise and fall times of 2 ms, and a plateau of 20 ms. When we measured the amplitude of CM, we set the amplitude of CM at 100 dB nHL before the salicylate microinfusion as a unit, and the amplitudes of CM after salicylate microinfusion were regarded as the ratios to that before the salicylate microinfusion. The compound action potential (CAP) was evoked by a click using a filter bandpass of 100−3 000 Hz. Stimulus intensities ranged from 10 to 100 dB nHL. Statistic Data Analysis. Levels of perilymph ascorbate were reported as a percentage of the basal level measured before salicylate microinfusion. A two-way repeated-measures ANOVA was used to analyze the effects of salicylate on the perilymph ascorbate levels. Two-way ANOVA is an appropriate analysis method for the study with a quantitative outcome and two (or more) categorical explanatory variables. In this study, one source of variation is due to the microinfusion of salicylate or perilymph (i.e., treatment group). The second source of differences lies in the time employed for recording the change of ascorbate in the cochlear of guinea pigs, and the third one originates from the interaction between the treatment group and the time. Therefore, the statistic analysis of the ascorbate results was performed using a two-way (group and time, with repeated measures on the latter factor) ANOVA to test the effects of treatment group, time, and their interactions. Individual time-point values between two groups were compared with the independent sample t-test. The accepted level of significance was 0.05, two-tailed. The amplitude of CM and threshold of CAP after salicylate microinfusion were compared with the basal levels (i.e., before salicylate microinfusion) with paired sample Student’s t test (P < 0.05).

Figure 1. Typical cyclic voltammogram obtained at the integrated capillary-electrode with the MWNT-modified CFME working electrode in artificial perilymph containing 5 mM ascorbate. Scan rate: 10 mV s−1. Inset, current−time response recorded for successive addition of ascorbate into artificial perilymph at the capillary-electrode at 0.05 V. The concentrations of ascorbate used for the first three and other additions were 1 μM and 5 μM, respectively.

still achieved in the artificial perilymph. Such a property successfully enables the selective in vivo measurements of ascorbate in the cochlear in this study. When the capillaryelectrodes were polarized at +0.05 V, well-defined steady-state current responses were recorded with the successive addition of ascorbate into the artificial perilymph (inset, Figure 1), demonstrating that the integrated capillary-electrodes prepared here were very responsive toward ascorbate. The current response was linear with ascorbate concentration within a concentration range from 1 μM to 50 μM (I (nA) = 0.048CAA (μM) − 0.039, γ = 0.999). As demonstrated in our earlier studies,14a,b,e the uses of carbon nanotubes as electrode materials could well avoid the interference from other kinds of electroactive species in the cerebral systems such as dopamine, 3,4-dihydroxyphenylacetic acid, uric acid, and 5-hydroxytryptamine, validating selective measurements of ascorbate in rat brain. Considering the complicated chemical nature in the cochlear of guinea pigs, we further in vivo investigated the selectivity of this method by microinfusing ascorbate oxidase (AAox) into the cochlear perilymph. As depicted in Figure 2, prior to local microinfusion of AAox, the capillary-electrode shows well-defined current response in the cochlear perilymph. The current response recorded was almost decreased to the background level of the electrode (Figure 1, inset) after exogenous infusion of 39.3 unit mL−1 AAox into the cochlea for 5 min at a rate of 2 μL min−1. This result substantially validates selective in vivo monitoring of ascorbate in the cochlear perilymph of guinea pigs with the integrated capillaryelectrodes with MWNTs as electrode materials. The basal level of the cochlear perilymph ascorbate was determined to be 45.0 ± 5.1 μM (n = 6). In Vivo Monitoring of Perilymph Ascorbate following Salicylate-Induced Tinnitus. Prior to in vivo monitoring of ascorbate in the cochlear perilymph of guinea pigs, electrocochleography was studied to verify the establishment of tinnitus models by microinfusion of salicylate into the cochlea in this study, based on the measurements of the changes in the amplitude of cochlear microphonic potential (CM) and the threshold of compound action potentials (CAP). As depicted in Figure 3A,B, with local microinfusion of 10 mM salicylate into

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RESULTS AND DISCUSSION Ascorbate Response at the Integrated CapillaryElectrodes. Figure 1 depicts typical cyclic voltammogram for ascorbate oxidation at the capillary-electrodes with the MWNTmodified CFME as working electrode in the artificial perilymph. The voltammogram reaches a well-defined steady state at 0.0 V with a half-peak potential of −0.04 V, which was quite similar to those obtained in the artificial cerebrospinal fluid (aCSF) and in the phosphate buffer,14a,b,e revealing that the accelerated electron transfer of ascorbate at MWNTs was 5435

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perilymph did not induce clear changes of the CAP threshold but that of 10 mM salicylate into cochlea significantly elevated the CAP threshold, reflecting that salicylate indeed induced auditory sensori-neural alterations in the inner ear. The experiments on the CM and CAP recordings demonstrate the establishment of tinnitus models by microinfusion of salicylate into the cochlea of guinea pigs.15 On the basis of the electrophysiological investigations, we implanted the capillary-electrodes into the cochlear perilymph of guinea pigs to in vivo monitoring of the changes of ascorbate level in the cochlear perilymph during the period of tinnitus induced by salicylate. Figure 4 displays a typical current Figure 2. Typical current−time response recorded at the integrated capillary-electrode in the cochlear perilymph of guinea pigs before and after local microinfusion of AAox (39.3 unit mL−1) through the capillary for 5 min at a rate of 2 μL min−1. The electrode was polarized at 0.05 V.

Figure 4. Typical current−time response recorded at the integrated capillary-electrodes in the cochlear perilymph of guinea pigs after local microinfusion of salicylate (red curve) and artificial perilymph (black curve) through the capillary. Blue curve represents the current−time response recorded in vitro with the capillary-electrodes in artificial perilymph with the addition of 10 mM salicylate. The electrode was polarized at 0.05 V.

response recorded with the capillary-electrodes with the microinfusion of salicylate from the capillary. For comparison, the current response with the microinfusion of artificial perilymph was also recorded. As shown in Figure 4, the microinfusion of salicylate results in a clear decrease in the current response (red curve), whereas that of artificial perilymph does not (black curve). Moreover, in vitro electrochemical experiments with the integrated capillaryelectrodes reveal that the addition of salicylate into the artificial perilymph did not change the current response (blue curve), suggesting that the salicylate-induced current increase (red curve) was not ascribed to the electrochemical oxidation of salicylate onto the MWNT-modified CFMEs. These results, along with the electrocochleographic studies mentioned above, suggest the ascorbate level in the cochlear perilymph of guinea pigs was remarkably decreased following the salicylate-induced tinnitus. The obtained current responses for ascorbate were converted into ascorbate concentration in the cochlear perilymph through the calibration curve mentioned above. As shown in Figure 5, the microinfusion of 10 mM salicylate into the cochlear (1 μL/ min, 5 min) significantly decreases the ascorbate level, reaching 28 ± 10% of the basal level (n = 6) (black curve). In contrast, the microinfusion of artificial perilymph (1 μL/min, 5 min) does not induce clear changes (n = 6) (red curve). Two-way ANOVA with repeated measures indicates a significant effect of the group (F(1,10) = 15.33, P = 0.0333), time (F(3,30) =

Figure 3. Typical (A,C) and statistical (B,D) results on the changes in the CM amplitude at 100 dB nHL in response to a 4 kHz tone burst (A,B) and in the CAP threshold (C,D) before and 5 min after infusion of salicylate into the cochlear. Asterisks indicate a significant difference (P < 0.05).

the cochlea through a micropump at the rate of 1 μL/min for 5 min, the amplitude of CM at 100 dB nHL in response to 4 kHz tone burst declines from unit to 0.743 ± 0.054 (P < 0.05), while CM evoked at 50 to 90 dB nHL remains unchanged (data not shown). On the other hand, the threshold of CAP evoked by a click ascends from 32.5 ± 3.51 dB to 62.00 ± 3.43 dB (P < 0.05) (Figure 3C,D). In the control experiments, the microinfusion of artificial perilymph did not induce significant changes in either amplitude of CM or the threshold of CAP (P > 0.05) (data not shown). It has been reported that CM and CAP are good electrophysiological tools to record the activity of the outer hair cells (OHCs) and auditory nerve fibers, respectively, providing an easy way to test the responses of OHCs and auditory nerve fibers to salicylate microinfusion in guinea pigs.15 In this study, the intracochlear administration of 10 mM salicylate (1 μL/min, 5 min) significantly decreased the amplitude of CM in response to a 4 kHz tone burst at 100 dB nHL, while that of artificial perilymph did not, indicating that there is acute ototoxicity of salicylate on OHCs to some extent. On the other hand, we found that the microinfusion of artificial 5436

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ACKNOWLEDGMENTS This research is financially supported by the NSF of China (Grant Nos. 20975104, 20935005, 21127901 to L. M., Grant 91132708 to P.Y., Grant 20905071 to Y.L.), the National Basic Research Program of China (973 Program, Grant 2010CB33502), Beijing Science Foundation of China (Grant 7112144, 7082100), and the Chinese Academy of Sciences (Grants KJCX2-YW-W25 and Y2010015).

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Figure 5. Statistical results of the change of the cochlear ascorbate level before and after microinfusion of salicylate (black curve) or the artificial perilymph (red curve) into the cochlear perilymph of guinea pigs. Asterisks indicate a significant difference compared with microinfusion of artificial perilymph with t test (P < 0.05). Arrow indicates the microinfusion of salicylate (black curve) or the artificial perilymph (red curve).

11.365, P < 0.000 001), and the interaction between time and group (F(3,30) = 9.478, P < 0.000 001). The independent sample t test following the two-way ANOVA further demonstrates that the level of ascorbate in the perilymph at point of 5 and 7 min after microinfusion of salicylate has a significant difference between two groups (P < 0.05). These results strongly suggest that the decrease of ascorbate in the cochlear may be involved in the mechanisms of tinnitus induced by salicylate. The decrease of ascorbate level in the perilymph following tinnitus induced by salicylate observed in our study was presumably considered to be associated, at least in part, with the protective role of ascorbate, implying endogenous mechanisms to control the toxic effects of salicylate on the cochlea.7,8,11,12,16 While further investigations on, for example, the relationship between ascorbate and other neurotransmitters such as glutamate and on the characteristics of ascorbate changes from the cochlea to the central auditory system remain to be conducted in our future studies, the results obtained with the method demonstrated in this study will be useful for understanding the intrinsic role of ascorbate in the mechanisms of tinnitus induced by salicylate.

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CONCLUSIONS We have successfully developed an electrochemical method for in vivo monitoring of ascorbate in the microenvironment of the cochlear perilymph of guinea pigs during salicylate-induced tinnitus by combining electrodes onto a glassy capillary to form integrated capillary-electrodes. The method is selective and could be used for ascorbate monitoring in the cochlear perilymph. This study essentially offers a new method for in vivo monitoring of the cochlear perilymph ascorbate following the salicylate-induced tinnitus, and the strategy demonstrated here can potentially extended for real-time monitoring of other kinds of neurochemicals involved in inner ear diseases. The results obtained in this study would be useful for investigation on chemical essences involved in tinnitus.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-010-62646525. Fax: +86-10-62559373. E-mail: [email protected] (L.M.); [email protected] (F.M.). Notes

The authors declare no competing financial interest. 5437

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